Neuroprotection and myelin repair using Nestorone®

ABSTRACT

Methods for treating neurodegeneration and/or myelination in patients are disclosed comprising treating the patient with a progestin compound which exerts binding to progesterone receptors and elicits progesterone-receptor-induced biological responses without interacting with the androgen receptor and without inducing androgen or glucocorticoid biological responses at a dosage sufficient to prevent or reduce neurodegeneration. The progestin compound preferably comprises 16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione, and the methods include combining the progestin compound with an estrogen compound to provide both contraception and treatment for myelin repair and neurodegeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national phase entry under 35 U.S.C. § 371of International Application No. PCT/US2010/053201 filed Oct. 19, 2010,published in English, which claims priority from U.S. Provisional PatentApplication No. 61/279,320 filed Oct. 19, 2009, all of which are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of prevention of myelindegeneration and neurodegeneration. More particularly, the presentinvention relates to the prevention or treatment of degenerative aspectsof diseases such as Multiple Sclerosis (MS), Alzheimer's Disease (AD)and Parkinson's Disease (PD), as well as for stroke.

BACKGROUND OF THE INVENTION

Multiple Sclerosis (MS) is a progressive and disabling disease of thecentral nervous system (CNS) affecting more than twice as many women asmen (1-4). Evidence suggests that neuronal damage begins early in MS(5), with acute axonal injury already present during activedemyelination. However, remyelination is known to occur in MS (6,7)where it protects against axon loss (8). Indeed, no significant axonaldamage can be observed in remyelinated plaques (5). Axons become lessreceptive to remyelination as MS progresses. Furthermore, a stroke is acerebrovascular incident which also leads to neuronal damage. In anexperimental model of transient middle cerebral artery occlusion (MCAO)the infarct volume induced by the occlusion was much larger in micedeprived of progesterone receptor (PR knockout mice) than in controlintact mice.

Neuronal damage can also occur in other contexts, such as with a stroke.A stroke is a cerebral vascular incident which results from aninterruption in the blood supply to brain cells. Neurons thus can bedestroyed because of their sensitivity to oxygen and glucosedeprivation, as well as from progressive spreading of nervous tissuedamage from an infarct site. There have thus been serious efforts totreat stroke patients to both protect neurons from being destroyed andavoid the spreading of lesions as well as to support regeneration ofdamaged tissue. Progesterone has previously been identified as anefficient neuroprotective agent. Indeed, progesterone itself is producedat increased rate in brain cells after lesion occurs. Progesteronetreatment has also been found to be effective in reducing lesion sizefollowing cerebral ischemia in animal models of stroke (18) and has beenfound to inhibit ischemic brain injury after brain artery occlusion(19).

Progestins such as Nestorone® have been found to exert proliferative andneuroprotective effects in the brain (20,21).

Approximately two-thirds of patients with relapsing-remitting MS arewomen of reproductive age.(9) It is known that a high level of femalesex steroids, such as that which occurs during pregnancy, may beresponsible for the remission of symptoms in women with MS. This isespecially true during the third trimester when estrogen andprogesterone (PROG) levels peak, while the relapse rate increases in thepost-partum period.(9)

Women with MS experience changes in their MS symptoms related topregnancy, the postpartum period, or menopause. In a study conducted inSweden (10),: 40% of the 148 women with MS who were interviewed reportedworsening of MS symptoms related to menopause, and more than a fourth ofthe younger women reported decreased symptoms during pregnancy. Everythird woman reported increased symptoms after delivery, suggesting thatthe sex steroids play a role in the protection (when present in highlevels during pregnancy) or worsening of the disease (when they decreaseafter delivery or at menopause).

An effective treatment strategy for conditions such as MS must alsoinclude therapeutic agents that reverse axon demyelination in order toprevent irreversible axon loss. Estrogen and progesterone, female sexhormones, may have beneficial effects on MS and neuroprotection.

The neurodegenerative process of several CNS diseases, includingMultiple Sclerosis (MS), Alzheimer's Disease (AD) and Parkinson'sDiseases (PD) involve neuroinflamation as well as neurodegeneration, andtheir frequency increases in women after menopause. Similarneurodegenerative processes are also present in stroke patients, orthose who have suffered the effects of a cerebrovascular incident.

In primary hippocampal neuron cultures treated with 17β-E2 andprogestins, alone and in combination, 48 hours before glutamate insult,estradiol, progesterone, and 19-norprogesterone, alone or incombination, protected against glutamate toxicity. In contrast,medroxyprogesterone acetate (MPA) failed to protect against glutamatetoxicity. Not only was MPA an ineffective neuroprotectant, but itattenuated the estrogen-induced neuroprotection when coadministered(11). These results may have important implications for the maintenanceof neuronal function during menopause and aging and for protectionagainst neurodegenerative diseases such as Alzheimer's disease byselecting the appropriate molecules for hormone therapy

Progesterone receptor (PR) expression and regulation of neuralprogenitor cell proliferation was investigated using NPC derived fromadult rat brain. Progesterone mediated neural progenitor cell (NPC)proliferation and concomitant regulation of mitotic cell cycle genes isa potential novel therapeutic target for promoting neurogenesis in themammalian brain (12).

SUMMARY OF THE INVENTION

In accordance with the present invention, these and other objects havenow been realized by the discovery of a method for treatingneurodegeneration in a patient comprising treating the patient with apharmaceutically effective dosage of a progestin compound which exertsbinding to progesterone receptors and elicitsprogesterone-receptor-induced biological responses without interactingwith the androgen receptor and without inducing androgen orglucocorticoid biological responses, the pharmaceutically accepteddosage being 5 mg/day or less, whereby neurodegeneration is prevented orreduced. In a preferred embodiment, the pharmaceutically effectivedosage of Nestorone® comprises from 100 to 450 μg/day. Preferably, thepharmaceutically effective dosage comprises a continuous dosage providedto the patient. In another embodiment, however, the pharmaceuticallyeffective dosage comprises an interrupted dosage provided to thepatient. Preferably, the interrupted dosage comprises three weeks on thedosage followed by one week off the dosage.

In accordance with one embodiment of the method of the presentinvention, the methods includes simultaneously treating the patient withan estrogen compound. Preferably, the estrogen compounds comprisesestradiol, and in a preferred embodiment the estradiol comprises fromabout 10 to 150 μg/day.

In accordance with another embodiment of the method of the presentinvention, the pharmaceutically effective dosage of the progestincompound comprises a transdermal dosage form.

In accordance with another embodiment of the method of the presentinvention, the progestin compound is selected from the group consistingof Nestorone®, 18-methyl Nestorone®, nomegestrol acetate, trimegestone,norgestimate, dienogest, drospirenone, chlormadinone acetate,promegestone, retroprogesterone, and 17-hydroxyprogesterone. In apreferred embodiment, the progestin compound comprises nomegestrolacetate, and the pharmaceutically effective dosage comprises from 2.5 to5 mg/day. In another embodiment, the progestin compound comprisestrimegestone, and the pharmaceutically effective dosage comprises fromabout 0.5 to 1 mg/day. In accordance with another embodiment, theprogestin compound comprises dienogest, and the pharmaceuticallyeffective dosage comprises from about 2 to 3 mg/day. In accordance withanother embodiment, the progestin compound comprises drospirenone, andthe pharmaceutically effective dosage comprises about 3 mg/day. Inanother embodiment, the progestin compound comprises chlormadinoneacetate, and the pharmaceutically acceptable dosage comprises about 5mg/day.

In accordance with another embodiment of the method of the presentinvention, the treating comprises providing the predetermined dosage ina transdermal form selected from the group consisting of transdermalgels, transdermal solutions, transdermal sprays, and transdermalpatches. In another embodiment, the method comprises providing thepredetermined dosage in a transdermal form selected from the groupconsisting of intravaginal tablets, intravaginal gels, and intravaginalrings.

In accordance with another embodiment of the method of the presentinvention, the method includes treating comprising a subcutaneousimplant.

In accordance with the present invention, a method is provided fortreating neurodegeneration in post-menopausal women comprising treatingthe post-menopausal women with a pharmaceutically effective dosage of aprogestin compound which exerts binding to progesterone receptors andelicits progesterone-receptor-induced biological responses withoutinteracting with the androgen receptor and without inducing androgenicor glucocorticoid biological responses at a dosage sufficient to preventor reduce neurodegeneration in the post-menopausal women, andsimultaneously providing a predetermined dosage of natural estradiol Inthe preferred embodiment of this method of the present invention, theprogestin compound comprises Nestorone®, and preferably is provided inan amount of between about 100 and 450 μg/day. In a preferredembodiment, the estradiol is provided in amounts of from about 10 to 150μg/day.

In one embodiment of this method of the present invention, thepharmaceutically effective dosage of the progestin compound comprises atransdermal dosage form selected from the group consisting oftransdermal gels, transdermal solutions, transdermal sprays, transdermalpatches, intravaginal tablets, intravaginal gels, and intravaginalrings.

In accordance with the present invention, a method is provided fortreating neurodegeneration exhibited in a condition selected from thegroup consisting of multiple sclerosis, Alzheimer's disease, Parkinson'sdisease, and stroke in a patient comprising treating the patient with apharmaceutically effective dosage of a progestin compound which exertsbinding to progesterone receptors and elicits progesteronereceptor-induced biological responses with interacting with the androgenreceptor and without inducing androgenic or glucocorticoid biologicalresponses wherein the pharmaceutically effective dosage comprises 5mg/day or less, whereby neurodegeneration is prevented or reducedthereby. Preferably, the progestin compound comprises Nestorone®. Inanother embodiment, the progestin compound is selected from the groupconsisting of 18-methyl Nestorone®, nomegestrol acetate, trimegestone,norgestimate, dienogest, drospirenone, chlormadinone acetate,promegestone, retroprogesterone, and 17-hydroxyprogesterone.

In accordance with the present invention, preliminary studies in tissueculture and animal models have shown that a particular class ofprogestin compounds, which includes Nestorone® (NES), a syntheticprogestin derived from 19-norprogesterone, with no androgenic,estrogenic, or glucocorticoid actions, have been shown to have greaterbeneficial effects on remyelination in in vitro models as compared, forexample, to progesterone, as well as to certain other progestincompounds. There are bioassays comparing the effects of differentprogestins. Nestorone® has no androgenic or estrogenic action at all andalso does not elicit a glucocorticoid effect except at doses 2,000-foldthe therapeutic dose. The other progestins such as levonorgestrel andMPA induce androgenic responses, MPA induces both androgenic andglucocorticoid responses, and norethyndrel and norethisterone exert withandrogenic and estrogenic responses. Furthermore, in recent studies, NEShas also been shown to stimulate proliferation of neural progenitorcells, again even higher than progesterone itself. These results haveled to the discovery of a method for treating neurodegeneration in apatient comprising treating the patient with a predetermined dosage of aprogestin compound which exerts binding to progesterone receptors andelicits progesterone-receptor-induced biological responses withoutinteracting with the androgen receptor and without inducing anyandrogenic or glucocorticoid biological responses at a dosage sufficientto prevent or reduce neurodegeneration. In connection with thisembodiment, the patient can comprise a male or female patient. Inaddition, in accordance with the present invention, a method fortreating neurodegeneration in a patient comprises treating the patientwith a pharmaceutically effective dosage of a progestin compound whichexerts binding to progesterone receptors and elicitsprogesterone-receptor-induced biological responses without interactingwith the androgen receptor and without inducing any androgenic orglucocorticoid biological responses at a dosage sufficient to prevent orreduce neurodegeneration, in conjunction with an estrogen compound. Inthis embodiment, the patient is preferably a female patient. Thespecified progestin compounds of this invention can be applied invarious ways, both orally and non-orally, including gels, patches,vaginal rings for women, or the like, in a wide range of dosages,ranging broadly from as low as about 30 μg/day absorbed by the patient,such as by the use of implants or the like, up to about 5 mg/day, suchas by the use of tablets or other such means of up to 3 to 5 mg.Similarly, in the case of estrogens, the amount delivered can range fromas low as 1 up to about 2,000 μg per day. It is also believed that atreatment for menopausal therapy including daily doses of NES betweenabout 100 and 400 μg/day, along with an estrogen, specifically estradiolin amounts of from 50 to 150 μg/day in gel formulations, will result inunexpectedly improved prevention or reduction in neurodegenerationand/or in myelin degeneration. This treatment can also be carried outvaginally, such as by use of a vaginal ring containing thesecompositions. The delivery can be either continuous or sequential, suchas sequential delivery of three weeks continuous delivery followed byone week of cessation of delivery.

It has further been discovered that these treatments can not only beapplied to post-menopausal women, but can be useful for the treatment inpreventing or reducing neurodegeneration in conditions such as MS, AD,PD, and in connection with stroke.

The primary focus of the present invention relates to methods fortreating neurodegeneration or myelin degeneration in patients, both maleand female. This primarily comprises treating these patients withpharmaceutically effective dosages of specific progestins such asNestorone®, at dosage levels of 10 to 450 μg/day and up to 5 mg/day orless in order to prevent or reduce neurodegeneration.

In one embodiment of the present invention, however, the invention isdirected specifically towards female patients. In one aspect of thistreatment, the progestin is combined with an estrogen compound, such asestradiol, so that in general both prevention or reduction inneurodegeneration and and/or myelin repair is effected along with eithercontraception or hormone therapy. Thus, in connection with youngpre-menopausal women of fertile age, with or without neurodegenerativeconditions such as MS, AD, PD, stroke, or the like, contraception isensured, while in post-menopausal women, again with or without theseneurodegenerative disorders, hormone therapy treatment can also beeffected. Thus, in addition to contraception and/or hormone therapytreatments, these combinations of compositions can be used to prevent orreduce relapses in MS in women either of reproductive age orpost-menopausally or during the post-partum period.

In a preferred embodiment, this is accomplished by administering aprogestin, such as those discussed above, preferably Nestorone®, andmost preferably in the form of a vaginal ring to administer thiscomposition in the form of the specific daily doses discussed above.

In the case of post-menopausal women, in one embodiment of the presentinvention, compounds of this invention are administered in the form of atransdermal gel, once again preferably including the combination of boththe progestin, such as Nestorone®, and estradiol. It is thus anticipatedthat this method can prevent or treat neurodegeneration in clinicalsituations of these medical conditions such as MS, AD, PD, and stroke.Preferably, the daily doses of the progestin, such as Nestorone®, willrange from 100 to 450 μg/day, again with or without associated estrogentherapy in such post-menopausal women. The progestin dosages can beadministered either continuously or interrupted by sequences of notreatment in order to allow for full efficacy in neuroproliferation andto induce endrometrial shedding.

On the other hand, in connection with the treatment of youngpre-menopausal women of fertile age, in one embodiment the presentinvention provides continuous long-term administration of the progestin,such as Nestorone®, at daily dosage rates of about 200 μg/day,preferably in the form of a vaginal ring. Again, this both insurescontraception with or without treatment of neurodegenerative conditionssuch as MS and the like. Furthermore, in view of the potentanti-ovulatory action of compounds such as Nestorone® itself, thelong-term administration of these dosages is adapted to preventpregnancies as well as to prevent relapses from MS. Thus, in accordancewith this invention, a new contraceptive agent is disclosed which hasadditional health benefits as opposed to all of the currentestrogen-progestin contraceptives which do not contain these progestinswith neuroprotective properties to be used in most women.

In accordance with a preferred embodiment of one aspect of the presentinvention, a composition is provided which includes a daily dose ofNestorone® for transdermal application, preferably in the form of a gel,containing between about 1 mg and 4.5 mg of transdermally appliedNestorone® (absorption of 10% resulting in about 100 to 450 μg/day ofNestorone®) which can be given alone, or which can be combined,preferably before use, for menopausal therapy, with estradiol,transdermally applied at from 0.5 to 1.5 mg, or 50 to 150 μg/day. In apreferred embodiment in which a vaginal ring is employed, the daily doseof Nestorone® is between about 100 and 300 μg/day either alone, or incombination with estradiol, preferably at doses of between 10 and 50μg/day. In this embodiment these dosages can be applied eithercontinuously, or sequentially, such as on a regimen of three weeks onand one week off.

In accordance with another embodiment of the present invention,post-menopausal women, with or without neurodegenerative disorders, canbe treated to induce neural progenitor cell proliferation by providingdaily dosage units comprising the progestins discussed above, includingNestorone®, in dosage amounts sufficient to induce neural progenitorcell proliferation.

The present invention also clearly has a general application for bothmales and females specifically for treating neurodegeneration or myelindegeneration in a patient. This method thus includes treating thepatient with a pharmaceutically effective dosage, preferably 5 mg/day orless, of the progestins of the present invention, preferably Nestorone®,so as to prevent or reduce neurodegeneration. In the preferredembodiment, the amount of Nestorone® utilized will constitute a dailydose of from between 100 to 300 μg/day, preferably about 200 μg/day.

The method of administering these doses of progestins, such asNestorone®, for example, can comprise non-oral administration. Non-oraladministration can include transdermal administration by means of gels,sprays, transdermal patches, or in the form of vaginal rings orimplants. Oral administration of the progestins of the present inventionwhich are orally active, can take place in the form of tablets,capsules, cachets, dragées, pills, pellets, granules, powder, solutions,emulsions, suspensions, and the like.

As for the specific progestin compounds which can be used in accordancewith this invention, these can include progestins such as Nestorone®, aswell as 18-methyl Nestorone®, nomegestrol acetate, trimegestone, as wellas non-androgenic progestins, such as norgestimate, dienogest,drospirenone, chlormadinone acetate, promegestone, retroprogesterone,and 17-hydroxyprogesterone. In general, the method of the presentinvention can thus be utilized for the prevention or reduction ofneurodegeneration and/or for myelin degeneration, and/or for thetreatment of conditions such as MS, AD, PD, or stroke.

As discussed above, the daily dose of the progestins in accordance withthe present invention is selected in order to exert binding toprogesterone receptors and to elicit progesterone-induced biologicalresponses without inducing either androgenic or glucocorticoidbiological responses.

In accordance with a preferred embodiment of the present invention, anew contraceptive agent is provided with additional health benefits, asopposed to all current estro-progestin contraceptives, which do notcontain such progestin with neuroprotective properties, to be used inmost women.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation comparing Nestorone® withprogesterone and promoting dose-dependent myelination;

FIG. 2 is a graphical representation comparing myelination withNestorone® lysolecithin, and RU486;

FIG. 3 is a graphical representation of the effect of progestins on theproliferation of progenitor cells;

FIG. 4 is a graphical representation of various progestins on theproliferation of progenitor cells;

FIG. 5 is a graphical representation of various progestins regardingneuroprotective efficacy;

FIG. 6A is a graphical representation comparing the NPC regeneration ofnorgestimate with progesterone;

FIG. 6B is a graphical representation comparing NPC regeneration withNestorone® compared to progesterone;

FIG. 6C is a graphical representation comparing NPC regeneration fornorethynodrel compared to progesterone;

FIG. 6D is a graphical representation comparing NPC regeneration fornorethindrone compared to progesterone;

FIG. 7A is a graphical representation comparing neuroprotective efficacyfor Nestorone® with progesterone;

FIG. 7B is a graphical representation comparing neuroprotective efficacyfor norethynodrel with progesterone;

FIG. 7C is a graphical representation comparing neuroprotective efficacyfor levonorgestrel with progesterone;

FIG. 7D is a graphical representation comparing neuroprotective efficacyfor norgestimate with progesterone;

FIG. 7E is a graphical representation comparing neuroprotective efficacyfor medroxyprogesterone acetate with progesterone;

FIG. 7F is a graphical representation comparing neuroprotective efficacyfor norethindrone with progesterone;

FIG. 8A is a graphical representation comparing NPC proliferation incell viability with various progestins;

FIG. 8B is a graphical representation comparing NPC proliferation incell viability with various progestins;

FIG. 8C is a graphical representation comparing NPC proliferation incell viability with various progestins;

FIG. 9A is a graphical representation of PCNA expression of variousprogestins; and

FIG. 9B is a graphical representation of percent of TUNEL-positivecells.

DETAILED DESCRIPTION

The present invention is most particularly based upon the discovery ofthe particular properties of certain progestins. Most particularly,these progestin compounds exert binding to progesterone receptors andelicit progesterone-receptor-induced biological responses withoutinteracting with the androgen receptor and without inducing theirandrogenic or glucocorticoid biological responses at a dosage sufficientto prevent or reduce neurodegeneration, and which dosage is nevertheless5 mg/day or less. These progestins thus include Nestorone®, 18-methylNestorone®, nomegestrol acetate, trimegestone, norgestimate, dienogest,drospirenone, chlormadinone acetate, promegestone, retroprogesterone,and 17-hydroxyprogesterone. Thus, this class of progestins excludesprogesterone and levonorgestrel, which interacts with the androgenreceptor, and which require a dosage of greater than 5 mg/day, andgenerally up to 10 mg/day or more, for efficacy. The progestin compoundsof the present invention can also include progestin compounds whichexert binding to progesterone receptors and elicitprogesterone-receptor-induced biological responses without inducingtheir androgenic or glucocorticoid biological responses.

We have set forth herein some presently preferred dosages for theprogestins, such as Nestorone®, which is highly preferred for use inconnection with the present invention. It is, however, within the skillof those in the pharmaceutical art to determine with routineexperimentation what dosage of each of these progestins will be needed,depending on the particular route of administration, to deliver such aneffective dose. However, while there are such variations as set forthbelow, it has been found that all of these progestin compounds of thepresent invention can be effectively utilized at dosages of 5 mg/day orless, which is considerably less than effective dosages of compoundssuch as progesterone. It is understood that the dosage of each of theseprogestins compounds, such as Nestorone®, administered in vivo may bedependent on the age, sex, health and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe pharmaceutical effect desired. The ranges of effective dosesprovided herein are not intended to be limiting and represent preferreddose ranges with the overall lower dosage range of 5 mg/day or lesshereof. However, the most preferred dosages within that overall rangemay be tailored to the individual subject, as is understood anddeterminable by one skilled in the relevant art. See, e.g., Berkow etal., eds., The Merck Manual, 16^(th) Ed., Merck & Co., Rahway, N.J.(1992); Goodman et al., eds., Goodman and Gilman's The PharmacologicalBasis of Therapeutics, 8^(th) Ed., Pergamen Press Inc., Elmsford, N.Y.(1990); Katzung, Basic and Clinical Pharmacology, Appleton & Lang,Norwalk, Conn. (1992); Avery's Drug Treatment Principles and Practice ofClinical Pharmacology and Therapeutics, 3^(rd) Ed., ADIS Press Ltd.,Williams & Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology, Little,Brown & Co., Boston, Mass. (1985); Remington's Pharmaceutical Services,17^(th) ed., Alphonzo R. Genaro, Mack Publishing Company, Easton, Pa.(1985); which references are entirely incorporated here by referencethereto.

The dosages can be determined by a clinician using conventional doseescalation studies. It can be expected to be within the above preferredranges. Furthermore, while this discussion has specifically referred tothe highly significant progestin component of the present invention, itcan, of course, also apply with equal force to the estrogen componenthereof.

In addition, by the term “pharmaceutically effective” it is meant thatamount which is sufficient to effect the desired changes in the subject.The amount will vary depending upon such factors as the potency of theparticular drug, the desired therapeutic effect, and the time span forwhich the method of application is intended to provide treatment. Thoseskilled in the pharmaceutical arts will be able to determine both toxiclevels and the minimum effective doses of the drug in accordance withstandard procedures. For instance, a proper dosage form can be preparedby measuring the in vivo rate or elution of a given drug by standardanalytic techniques, e.g., spectroscopic or radioimmunoassay analysis.In vitro diffusion of the drug from a delivery device of the presentinvention may be determined, for example, by the methods disclosed inChien et al., J. Pharm. Sci., 63, 365 (1974) or by the methods describedin U.S. Pat. No. 3,710,795, the disclosures of which are incorporated byreference herein.

The applicants have discovered that these specific progestin compoundscan have unexpected properties in terms of their myelination and alsofor the treatment of neurodegeneration, and in particular treatment ofconditions such as MS, AD, PD, and stroke, and furthermore that theseunexpected properties can be obtained in conjunction with treatment, ofcontraceptive action with these compounds known to be useful for thatpurpose.

A particular preferred use of the progestin compounds of the presentinvention is thus in conjunction with an estrogen compound. By estrogencompound one of skill in this art will appreciate that the estrogen canbe selected from the group consisting of estradiol, ethinyl estradiol,estradiol sulfimates, estradiol valerate, estradiol acetate, estradiolbenzoate, estrone, estriol, estriol succinate, and conjugated estrogensincluding conjugated equine estrogens such as estrone sulfate,17β-estradiol sulfate, 17α-estradiol sulfate, equilin sulfate,17β-dihydroequilin sulfate, 17α-dihydroequilin sulfate, equileninesulfate, 17β-dihydroequilenine sulfate, 17α-dihydroequilenine sulfate,estetrol, or mixtures thereof. Most preferred is estradiol.

The combination of progestins with estrogens for contraceptive purposesis widely known. Indeed, since progestins alone cannot normally be usedwithout developing poor bleeding patterns in women, nor forpostmenopausal use, it has become necessary to combine these progestinswith estrogens for these purposes. Furthermore, while the primary thrustof the present invention is based upon the discovery that certainprogestins as described herein possess unexpectedly superior propertiesin connection with neuroprotection and myelination, and the addition ofan estrogen is not necessarily for assisting in that objective, it isalso possible that the use of certain estrogens in combination withthese progestins provides even greater unexpected results in terms ofneuroprotection, or neuroregeneration and myelin repair.

In experiments conducted in organotypic neonatal rat or mouse cerebellarslice culture, progesterone accelerated axon myelination (13,14). In astudy conducted in accordance with this invention, both progesterone(PROG) and Nestorone® (NES) were found to promote dose-dependentmyelination, as measured by myelin basic protein (MBP) immunoreactivity.NES was found to be significantly more potent than PROG, as NES at 20 μMwas as active as PROG at 50 μM (FIG. 1). It was also shown that theintracellular progesterone receptor (PR) may mediate the promyelinatingactions of PROG as the treatment did not increase myelination incerebellar slices from PR knockout mice.

In the same animal model, cerebellar slices were cultured untilmyelination was complete, then incubated overnight with lysolecithin(LYSO) to produce demyelination utilizing a known technique, (15)followed by 3 days of incubation with NES 20 μM in fresh culture medium(10% penetration in slices). Slices were immunostained for MBP. As shownin FIG. 1, NES produced remyelination. MBP staining intensity wasmeasured in cerebellar slices after completion of normal myelination,after demyelination with lysolecithin, and after 3 days of incubationwith NES at 20 μM, RU486 at 10 μM, or NES+RU486. NES-stimulatedremyelination of lysolecithin-demyelinated cerebellar slices may involvethe classic progesterone receptor (PR), as RU486 appeared to inhibit NESactivity in this model (FIG. 2).

According to the above described in vitro studies, it appears thatmyelination/remyelination action of NES may be mediated by theprogesterone receptors (PR). Thus, NES, which is a potent agonist of PRand one of the most potent progestins without androgenic activity thatinduces PR-related biological responses, appears to improve myelinregeneration even better than progesterone, and this can become atreatment of the diseases or conditions associated with demyelination.

In postmenopausal women, the increase in neurodegenerative diseases hasbeen related to the lack of estrogen and little attention has been paidto the role of progesterone. Study of the proliferation of neuronal stemcells in a rodent model showed that in the subventricular zone of thebrain these cell rapidly divide and give rise to neuroblasts that willbecome interneurons. Progesterone increases the proliferation of theseprogenitor cells. Among various progestins tested for cellproliferation, Nestorone® is as effective or even more effective thanprogesterone. Norethynodrel and norgestimate were also moreproliferative than progesterone (FIG. 3). However other progestins wereeither less effective than progesterone (NET, LNG) or antagonistic (MPA,NETA) on the proliferation (FIG. 4).

The effect of NES and other progestins on CNS plasticity and theneuroprotective efficacy against glutamate toxicity has also beenevaluated. LDH was measured, as a well-accepted assay, to determine sucheffect after exposure of the cells to glutamate, and the neuronalviability was assessed under the action of various progestins. FIG. 5shows that three progestins have comparable effect to progesterone andat 10-7M NES, and PROG exerted the higher efficacy while LNG, anandrogenic progestin, was more active than PROG at lower doses.

EXAMPLE 1

In vitro studies were carried out to determine neural progenitor cell(NPC) regeneration in rats. 5-Bromo-2-deoxiuridine (BrdU)chemiluminescence enzyme-linked immunosorbant assay (ELISA) and theresults are shown in FIGS. 6A-D herein. Cell proliferation wasdetermined by S phase incorporation of BrdU. After 4 to 6 hoursstarvation (medium without supplements), rNPCs were loaded with 10 μMBrdU in the presence or absence of bFGF and varying concentrations of P₄or test progestins in unsupplemented maintenance medium for 1d. TherNPCs were then processed as described previously (1, 14). Aftersubtracting the value of the blank (without BrdU loading), data wereanalyzed using a one-way ANOVA, followed by a Neuman-Keuls post hoctest. These results demonstrate that at 24 hours norgestimate was morepotent in cell proliferation than progesterone at all concentrations.Nestorone® and progesterone were comparably efficacious at their EC₁₀₀concentrations. Norethynodrel produced comparable effects toprogesterone at the low nanomolar range but was significantly moreefficacious than progesterone at high nanomolar ranges. Norethindronewas less effective than progesterone and levonorgestrel andnorethindrone acetate exerted minimal or no effect on proliferationwhile medroxyprogesterone acetate (MPA) significantly inhibitedproliferation at multiple concentrations.

EXAMPLE 2

Comparisons of neuroprotection against neurodegenerative insults werecarried out. Efficacy was determined in connection with the protectionof primary hippocampal neurons against degeneration induced by excitoxicglutamate. Hippocampal neuronal cultures grown on 90-well culture platesfor 7d in vitro were pretreated with vehicle alone or test compounds,followed by exposure to 200 μM glutamate as previously described (13).After glutamate exposure, cultures were washed with HEPES-bufferedsaline solution and replaced with fresh NBM containing the testcompounds or combinations. Cultures were returned to the incubator andincubated for 24 hours prior to analysis of neuronal viability usingcolorimetric LDH release in the media. Dose-response analysis wasconducted for each of the progestins tested, and the results are shownin FIGS. 7A-F. Neuroprotective efficiency was calculated as follows:NE=(V_(sample)−V_(glutamate))/(V_(subcontrol)−V_(glutamate)). As can beseen, Nestorone® induced comparable neuroprotective efficacy to that ofprogesterone. Norethyndrel was more potent than progesterone at minimaleffective concentrations while levonorgestrel showed comparable efficacyto progesterone. Norgestimate was found to be less potent thanprogesterone, as were both MPA and norethindrone.

EXAMPLE 3

The generalized ability of the above in vitro findings to the in vivocondition were investigated. Analyses of NPC proliferation in rats andcell viability were conducted in three-month-old Sprague-Dawleyovariectomized female rats with various of the progestins. Cell cycleprotein expression was determined by Western blot analysis, and theresults as shown in FIG. 8A indicated that Nestorone® appeared to beslightly superior to progesterone in terms of increased PCNA expressionat the protein level. Levonorgestrel and MPA, on the other hand, had nosignificant effect on PCNA expression, while CDC2 protein expression wassignificantly increased by both progesterone and Nestorone®, but notwith levonorgestrel and MPA. In order to assess the total number ofBrdU+ cells, the contralateral hippocampal hemisphere used for proteinanalysis was fixed and processed by FACS analysis. The total number ofBrdU+ cells per each hippocampus was determined and normalized to thatof vehicle control. The results are shown in FIG. 8B demonstrating thatNestorone® was slightly superior to progesterone in significantlyincreasing Brd+ cell numbers while levonorgestrel was comparable toprogesterone while MPA had no significant effect on cell proliferationin vivo. In order to measure cell viability in terms of promotion ofmitochondrial function and reduction in oxidative damage expression ofthe alpha subunit of ATP synthase-Complex V (CVα) of the mitochondrialoxidative phosphorylation pathway was assessed by Western blot analysis.The results as shown in FIG. 8C demonstrate that Nestorone® increasedCVA expression even greater than progesterone and levonorgestrel whileagain MPA exerted no significant effect on CVα expression levels.

EXAMPLE 4

The effects of various progestins on apoptosis was studied using Westernblot analysis to determine the expression level of Bax, an apoptosismediator by translation to the mitochondria to release apoptotic factorssuch as cytochrome c and Bcl-2. The ratio of Bax to Bcl-2 was used as anindicator of in vivo apoptotic activity. The results obtaineddemonstrated that both progesterone and Nestorone® had no effect on theratio of Bax/Bcl-2 expression, while levonorgestrel and MPAsignificantly increased the ratio demonstrating a pro-apoptotic effecttherein.

EXAMPLE 5

The impact of the combination of 17β-estradiol (E₂) and variousprogestins on neurogenesis and cell viability in vitro was also carriedout. Young adult ovariectomized female Sprague Dawley rates were thusdivided into six groups and received injection of either E₂ alone or E₂combined with one of the progestins. Hippocampi were isolated 24 hourslater for Western blot analysis and flow cytometry to determine theimpact of treatment on cell viability and neurogenesis respectively. Theexpression level of PCNA was assessed to determine the impact of thetreatment compounds on entry into the cell cycle required forneurogenesis. The results demonstrated that Nestorone® plus E₂ inducedthe greatest magnitude of PCNA expression and neural progenitor cellproliferation (see FIG. 9A).

Importantly, Nestorone®, in combination with estradiol, did not increaseneural progenitor cell death as evidenced by no increase in TUNELpositive cells, a marker for apoptosis. In contrast, both MPA andlevonogestrel, in combination with estradiol, significantly increasedapoptosis, as evidenced by an increase in TUNEL positive cells (see FIG.9B). As noted above, both MPA and levonorgestrel increased cellproliferation.

The present invention provides a method of stimulating neuroregenerationand possibly inhibiting and reversing neurodegenerative disorders suchas MS or AD, as well as stroke.

The proposed method comprises reversing the myelin degeneration with adose of NES in the range of about 100 to 450 μg per day administeredeither by a vaginal ring or in a vaginal gel alone or in associationwith estradiol.

In another embodiment of the invention, NES, or a progestin withoutandrogenic or glucocorticoid properties, is administered topostmenopausal women who receive low doses of estrogen as hormonetherapy and a possible prevention of neurodegeneration.

The present invention pertains to the discovery that NES is more activethan progesterone to stimulate progenitor neuronal cells as well as theregeneration of myelin. A further core aspect of the invention is thatagents capable of binding to the progesterone receptors and inducingPR-induced biological responses, would be effective in preventing orreversing neurodegeneration, for women in reproductive age as well aspostmenopausal, women .

The term “Nestorone®” (NES) refers to a 19-norprogesterone derivativethat exerts a potent progestational and antiovulatory action and doesnot carry androgenic or estrogenic or glucocorticoid actions attherapeutic levels (16). In particular, it refers to16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione, which wasformerly referred to as ST1435.

The term “DDU” herein refers to daily dosage units wherein the DDU is inoral formulation for other 19norprogesterone derivatives that are activeorally (not NES)., or in a vaginal (gel or ring) or transdermalformulation (gel, spray).

The term “contraceptive agent” used herein refers to medicationsadministered in order to prevent or reduce the likelihood of pregnancy.

The present invention is based on the fact that progesterone stimulatesmyelin repair. These effects are mediated by progesterone receptors(PR). The present invention reveals that NES is more active than PROGand can regenerate myelin at doses that also exert contraceptiveefficacy.

The present invention also reveals that progenitor cells of neuronaltissue proliferate when cultured with progesterone and moreover with NESat lower doses indicating a higher activity.

Also, progesterone and some progestins, especially NES and alsonorgestimate (a non-androgenic gonane) and norethynodrel (an estraneprogestin with estrogenic activity) stimulate progenitor cellproliferation.

Based on the superior effect of NES on myelin stimulation as well as onneuroregeneration, it is a purpose of the present invention to improvethe medical conditions of multiple sclerosis and neurodegenerativedisorders and at the same time providing a contraception in women offertile age or a hormonal therapy in women who are in the menopause.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

INDUSTRIAL APPLICABILITY

The methods of treating neurodegeneraton or mylenation which aredisclosed in this application are particularly useful in the form of anon-oral dosage form of a specified progestin, such as Nestorone®,either alone or in combination with an estrogen, such as estradiol. Thiscomposition is used in the form of a transdermal product, such as a gel,solution, spray or patch, or in the form of a vaginal ring, which canthus be used by a patient to reduce neurodegeneration and when combinedwith the estrogen, to also be used for contraception and/or hormonereplacement therapy.

REFERENCES

-   1. Confavreux C, Aimard G, Devic M. Course and prognosis of multiple    sclerosis assessed by the computerized data processing of 349    patients. Brain 1980; 103(2):281-300-   2. Confavreux C, Vukusic S. Natural history of multiple sclerosis: a    unifying concept. Brain 2006; 129(Pt 3):606-16-   3. Pugliatti M, Rosati G, Carton H, Riise T, Drulovic J, Vecsei L,    Milanov I. The epidemiology of multiple sclerosis in Europe. Eur. J.    Neurol. 2006; 13(7):700-22-   4. Dutta R, Trapp B D. Pathogenesis of axonal and neuronal damage in    multiple sclerosis. Neurology 2007; 68(22 Suppl 3):S22-S31-   5. Kornek B, Storch M K, Weissert R, Wallstroem E, Stefferl A,    Olsson T, Linington C, Schmidbauer M, Lassmann H. Multiple sclerosis    and chronic autoimmune encephalomyelitis: a comparative quantitative    study of axonal injury in active, inactive, and remyelinated    lesions. Am. J Pathol. 2000; 157(1):267-76-   6. Patani R, Balaratnam M, Vora A, Reynolds R. Remyelination can be    extensive in multiple sclerosis despite a long disease course.    Neuropathol. Appl. Neurobiol. 2007; 33(3):277-87-   7. Patrikios P, Stadelmann C, Kutzelnigg A, Rauschka H, Schmidbauer    M, Laursen H, Sorensen P S, Bruck W, Lucchinetti C, Lassmann H.    Remyelination is extensive in a subset of multiple sclerosis    patients. Brain 2006; 129(Pt 12):3165-72-   8. Irvine K A, Blakemore W F. Remyelination protects axons from    demyelination-associated axon degeneration. Brain 2008; 131(Pt    6):1464-77-   9. Confavreux C, Hutchinson M, Hours M M, Cortinovis-Tourniaire P,    Moreau T. Rate of pregnancy-related relapse in multiple sclerosis.    Pregnancy in Multiple Sclerosis Group. N. Engl. J. Med. 1998;    339(5):285-91-   10. Holmqvist P, Wallberg M, Hammar M, Landtblom A M, Brynhildsen J.    Symptoms of multiple sclerosis in women in relation to sex steroid    exposure. Maturitas. 2006 May 20; 54(2):149-53-   11. Nilsen J, Brinton R D. Impact of progestins on estrogen-induced    neuroprotection: synergy by progesterone and 19-norprogesterone and    antagonism by medroxyprogesterone acetate. Endocrinology 2002;    143(1):205-12-   12. Liu L, Wang J, Zhao L, Nilsen J, McClure K, Wong K, Brinton R D.    Progesterone increases rat neural progenitor cell cycle gene    expression and proliferation via extracellularly regulated kinase    and progesterone receptor membrane components 1 and 2.    Endocrinology. 2009 July; 150(7):3186-96.-   13. Ghoumari A M, Ibanez C, El Etr M, Leclerc P, Eychenne B,    O'Malley B W, Baulieu E E, Schumacher M. Progesterone and its    metabolites increase myelin basic protein expression in organotypic    slice cultures of rat cerebellum. J. Neurochem. 2003; 86(4):848-59-   14. Ghoumari A M, Baulieu E E, Schumacher M. Progesterone increases    oligodendroglial cell proliferation in rat cerebellar slice    cultures. Neuroscience 2005; 135(1):47-58-   15. Birgbauer E, Rao T S, Webb M. Lysolecithin induces demyelination    in vitro in a cerebellar slice culture system. J. Neurosci. Res.    2004; 78(2):157-66-   16. Kumar N, Koide S S, Tsong Y, Sundaram K. Nestorone: a progestin    with a unique pharmacological profile. Steroids 2000;    65(10-11):629-36-   17. Sitruk-Ware R. New progestagens for contraceptive use. Hum.    Reprod. Update. 2006; 12(2):169-78-   18. Gibson L, Gray L J, Bath P M, Murphy S P (2008) Progesterone for    the treatment of experimental brain injury; a systematic review.    Brain 131:318-328-   19. Sayeed I, Wali B, Stein D G (2007) Progesterone inhibits    ischemic brain injury in a rat model of permanent middle cerebral    artery occlusion. Restor. Neurosci. 24:151-159-   20. Brinton R D, Thompson R F, Foy M R, Baudry M, Wang J, Finch C E,    Morgan D E, Pike C J, Mack W J, Stanczyk F C, Nilsen J (2008)    Progesterone receptors: form and function in the brain. Front    Neuroendocrinal 29:313-339-   21. Wang J M, Liu L, Irwin R W, Chen S, Brinton R D (2008)    Regenerative potential of allopregnanolone. Brain Res. Rev.    57:398-409

The invention claimed is:
 1. A method for remyelination in a patientcomprising treating said patient with a pharmaceutically effectivedosage of a progestin compound which exerts binding to progesteronereceptors and elicits progesterone-receptor-induced biological responseswithout interacting with the androgen receptor and without inducingandrogen or glucocorticoid biological responses selected from the groupconsisting of 16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione,18-methyl Nestorone, nomegestrol acetate, trimegestone, andpromegestone, said pharmaceutically effective dosage being an effectiveamount for remyelination comprising 100 to 450 μg/day, wherebyneurodegeneration is reduced.
 2. The method of claim 1 includingsimultaneously treating said patient with an estrogen compound.
 3. Themethod of claim 1 wherein said estrogen compound comprises estradiol. 4.The method of 3 wherein the dosage of said estradiol comprises fromabout 10 to 150 μg/day.
 5. The method of claim 1 wherein saidpharmaceutically effective dosage comprises a continuous dosage providedto said patient.
 6. The method of claim 1 wherein said pharmaceuticallyeffective dosage comprises an interrupted dosage provided to saidpatient.
 7. The method of claim 6 wherein said interrupted dosagecomprises three weeks on said dosage followed by one week off saiddosage.
 8. The method of claim 1 wherein said pharmaceutically effectivedosage of said progestin compound comprises a transdermal dosage.
 9. Themethod of claim 1 wherein said treating comprises providing saidpredetermined dosage in a transdermal form.
 10. The method of claim 9wherein said transdermal form is selected from the group consisting oftransdermal gels, transdermal solutions, transdermal sprays, andtransdermal patches.
 11. The method of claim 9 wherein said method oftreating comprises a transdermal product selected from the groupconsisting of intravaginal tablets, intravaginal gels, and intravaginalrings.
 12. The method of claim 1 wherein said method of treatingcomprises a subcutaneous implant.
 13. The method of claim 1 wherein saidprogestin compound comprises16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione.
 14. A method forremyelination in post-menopausal women comprising treating saidpost-menopausal women with a pharmaceutically effective dosage of aprogestin compound which exerts binding to progesterone receptors andelicits progesterone-receptor-induced biological responses withoutinteracting with the androgen receptor and without inducing androgenicor glucocordicoid biological responses selected from the groupconsisting of 16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione,18-methyl Nestorone, nomegestrol acetate, trimegestone, and promegestoneat a dosage of an effective amount for remyelination in saidpost-menopausal women comprising 100 to 450 μg/day, and simultaneouslyproviding a predetermined dosage of natural estradiol.
 15. A method forremyelination exhibited in a condition selected from the groupconsisting of Multiple Sclerosis, Alzheimer's Disease, and Parkinson'sDisease in a patient comprising treating said patient with apharmaceutically effective dosage of a progestin compound which exertsbinding to progesterone receptors and elicitsprogesterone-receptor-induced biological responses selected from thegroup consisting of16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione, 18-methylNestorone, nomegestrol acetate, trimegestone, and promegestone withoutinteracting with the androgen receptor and without inducing androgenicor glucocorticoid biological responses at a dosage comprising 100 to 450μg/day as an effective amount for remyelination and sufficient to reducerelapses of said conditions.